Members of the Burkholderia species that cause diseases in humans include the Burkholderia cepacia complex (Bcc), which can cause acute and chronic infections in cystic fibrosis patients, and Burkholderia mallei and Burkholderia pseudomallei, which are category B select biothreat agents that are responsible for glanders and meliodiosis, respectively. Bcc are considered opportunistic pathogens affecting immunocompromised individuals, while B. mallei and B. pseudomallei are highly virulent organisms, and would pose serious health threats if intentionally released; there are currently no approved vaccines available for any of these bacteria. Our long-term objective is to develop effective vaccines for these infectious bacterial pathogens. The antigens that will serve as model vaccine targets in this 2-year exploratory grant are the O antigen portion of lipopolysaccharide synthesized by an epidemic strain of Bcc, and the sequenced B. mallei and B. pseudomallei strains. Our rationale is that these carbohydrate antigens are the outermost components of bacterial cells and may be immunodominant protective antigens. We will express these antigens on the surface of attenuated strains of Salmonella and deliver these vaccines to mice. This system should allow these O antigens to be in a more "native confirmation" as well as preclude the need to conjugate them to protein carriers in order to elicit a functional immune response. An additional advantage of this approach is that these vaccines are easy to administer, either orally or intranasally, in a non-invasive, needle-free manner. We will transfer and express the genes encoding the enzymes for the synthesis of these O antigens to attenuated Salmonella strains. Once O antigen expression is confirmed in this heterologous host (Specific Aim 1), these recombinant strains will be used to vaccinate mice by either the oral or intranasal route. After an immune response is generated (Specific Aim 2), these mice will be challenged using standard infection models for each pathogen to determine the protective efficacy of these vaccinations. For Bcc, we will use the agar bead model, and for B. mallei and B. pseudomallei we will infect the mice via the intranasal route (Specific Aim 3). We hypothesize that immunized mice will be protected with regard to bacterial burden, show increased time to death when this occurs, and less overall mortality compared to non-immune mice. If one route of immunization is more efficacious, future experiments will define how best to optimize this vaccine approach to elicit a protective immune response as well as to generate passive immunotherapeutic reagents. Our long- term goal is to exploit this knowledge and technology to produce effective vaccines for human use. The focus of this two-year exploratory grant is on the development of vaccines against the species of Burkholderia that cause infections in humans. In particular, we propose to express O antigens from an epidemic strain of Burkholderia cenocepacia, which is an emerging pathogen in cystic fibrosis patients, and the biothreat agents Burkholderia mallei and Burkholderia pseudomallei, on attenuated Salmonella strains. These constructs will be administered to mice and their efficacy tested in relevant mouse models of infection. Our long-term goal is to develop similar reagents for human use. [unreadable] [unreadable] [unreadable]